Oncogene EVI1 drives acute myeloid leukemia via a targetable interaction with CTBP2

Acute myeloid leukemia (AML) driven by the activation of EVI1 due to chromosome 3q26/MECOM rearrangements is incurable. Because transcription factors such as EVI1 are notoriously hard to target, insight into the mechanism by which EVI1 drives myeloid transformation could provide alternative avenues for therapy. Applying protein folding predictions combined with proteomics technologies, we demonstrate that interaction of EVI1 with CTBP1 and CTBP2 via a single PLDLS motif is indispensable for leukemic transformation. A 4× PLDLS repeat construct outcompetes binding of EVI1 to CTBP1 and CTBP2 and inhibits proliferation of 3q26/MECOM rearranged AML in vitro and in xenotransplant models. This proof-of-concept study opens the possibility to target one of the most incurable forms of AML with specific EVI1-CTBP inhibitors. This has important implications for other tumor types with aberrant expression of EVI1 and for cancers transformed by different CTBP-dependent oncogenic transcription factors.

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Figs. S1 to S7 Legend for table S1 Uncropped Western blots for panel S1A
Other Supplementary Material for this manuscript includes the following: EVI1 IP followed by CTBP2 Western blot (top panel) or vice versa (bottom panel) in MUTZ3 whole-cell lysates.Growth curve in SB1690CB with two independent shRNAs per gene for CTBP2 and MECOM.To determine significance, an exponential growth model (Y= logY0 + k*X)) is fit and best-of-fit value for growth rate k is compared between groups with an extra sum-of-squares F-test.The control cells are untransduced and shcontrol-transduced cells (n = 2 per group; Mean + SD plotted).Fig S2J.
Western blot of knock-down efficiency of SB1690CB growth curve depicted in panel S2I.Quantification of the heatmap in panel Fig.S3B.For each track, the normalised CTBP2 signal is plotted versus the normalised reads in the indicated ChIP-seq-tracks, for all CTBP2 peaks with a window of ±1000bp.Correlation coefficients and linear regression equations are shown for log10-transformed data with a pseudo count of 1.  Schematic outline of the MAPPIT assay to measure protein-protein interaction (here for EVI1 and CTBP2).The MAPPIT receptor is based on the EPO receptor, which harbors an intracellular domain that can be phosphorylated by JAK in normal JAK-STAT signalling (1).
In the modified MAPPIT receptor, the intracellular domain fused to EVI1 and the Tyr phosphorylation sites are mutated.Gp130 is fused to CTBP2 (2).Because EVI1-CTBP2 can interact with each other, CTBP2 is recruited to the EPO-EVI1 receptor.In this way, when signal is transmitted by ligand binding (2), JAK will phosphorylate tyrosines on CTBP2-gp130 to subsequently activate STAT3 (3).Phosphorylated STAT3 is transported into the nucleus where it will activate a STAT3 responsive luciferase reporter (4).

Fig S6B.
Volcano plot of differential CTBP2 and EVI1 binding (by ChIP-seq) analysis (DiffBind) in MUTZ3 cells that were transduced with dox-inducible 4xPLDLS expression.Doxinduced versus untreated bulk cell populations s are compared at 72hrs; n = 2 per group.
Regions from panel S6E are labelled in both plots.The number of peaks is indicated above the plots.The distribution of all PLDLS-target-gene-annotated-peaks is visualised above the axis.342 peaks could be annotated to a upregulated PLDLS target gene (88 unique genes in total, on average 3.9 peaks per gene) and 47 to a downregulated PLDLS target gene (18 unique genes in total, or 2.6 peaks per gene on average).Flow-cytometric analysis of MUTZ3 cells that were transduced with 4xPLDLS and 4X PLASS and subsequently mixed in a 50:50 ratio.Data is presented as a fraction of live cells.For Fig. 5D, the frequency of PLASS or PLDLS within the total transduced fraction is determined.

Fig S7C.
Flow-cytometric analysis of input and 5 mice transplanted with SB1690 with Empty vector-Emerald and Empty vector -mCherry mixed in a 1:1 ratio.

Fig S7D.
Flow-cytometric analysis of input and 7 mice transplanted with SB1690 with PLASS-Emerald and PLDLS -mCherry mixed in a 1:1 ratio.

Full
version of Fig 1B, including all the protein labels and groups of proteins removed because they are likely contaminants.

Figure S5 .
Figure S5.Supplement to Fig. 3 Fig S5A.Protein alignment of all PLDLS sites +-15 AA from the human proteome.Distance clustering is based on amino acid similarity (Fitch matrix).Fig S5B.Subcellular localisation of PLDLS containing proteins, whether they have been identified previously as CTBP1/2 interactors (Based on BioGrid) and protein expression from DIAquantified mass spectrometry nuclear lysate input samples.Fig S5C.ChimeraX interaction on AlphaFold predicted heterodimer models of CTBP2 with the PLDLS site from the indicated protein.The count refers to the number of predicted models where the interaction with this residue was found.
Fig S6C.Fold-change of CTBP2 peaks in response to 4x PLDLS treatment in MUTZ3.Peaks are grouped according to whether they overlap with EVI1 peaks (n = 4026), or not (n =1608).Fig S6D.Significantly differentially expressed (DE) genes (adj.P-value <0.05 and absolute foldchange > 2) upon 4x PLDLS treatment in MUTZ3 cells (n = 2, 72 hrs post-TD, 162 genes in total).The fraction of DE genes that are bound by CTBP2 are highlighted in dark.Peakto-gene annotation is based firstly on whether the peak directly overlaps with a putative regulatory element associated to gene expression, and if it does not overlap with such an element, to the nearest protein-coding gene.Fig S6E.CTBP2 peaks are ranked (x-axis) based on their enrichment over input (y-axis).The top-30 peaks annotated to genes from figure C based on highest CTBP2 binding are labelled.The distribution of all PLDLS-target-gene-annotated-peaks is visualised above the axis.342 peaks could be annotated to a upregulated PLDLS target gene (88 unique genes in total, on average 3.9 peaks per gene) and 47 to a downregulated PLDLS target gene (18 unique genes in total, or 2.6 peaks per gene on average).

Figure S7 .
Figure S7.Supplement to Figure 4/5 Fig S7A.Counts of polymorphic (differentiated) nuclei as illustrated by arrows in panel E.Significance is tested with one-way ANOVA with multiple comparisons (Empty vector as reference; mean + SD plotted)Fig S7B.Flow-cytometric analysis of MUTZ3 cells that were transduced with 4xPLDLS and 4X PLASS and subsequently mixed in a 50:50 ratio.Data is presented as a fraction of live cells.For Fig.5D, the frequency of PLASS or PLDLS within the total transduced fraction is determined.
Quandrants indicate single-positive and double-positive cells.